Influence of the genus Bacillus bacteria on the content of H2O2 and the activity of hydrolases and their inhibitors in potato plants during Phytophthora infestans Mont. de Bary infection

The authors studied the effect of treatment with bacteria Bacillus subtilis Cohn (strains 26D) and B. thuringiensis Berliner (strain B-6066) on the hydrogen peroxide (H2O2) content, the activity of hydrolytic enzymes and their protein inhibitors in potato plants (Solanum tuberosum L.) in connection with development of resistance to the late blight pathogen – oomycete Phytophthora infestans Mont. de Bary. Studies were carried out on potato plants of the susceptible Early Rose potato cultivar that were treated with a suspension of B. subtilis and B. thuringiensis bacteria (108 cells/ml) and infected with P. infestans (107 spores/ml). A decrease in the degree of leaf damage by oomycete was revealed under the influence of the genus Bacillus bacteria, depending on the strain. The increase in potato resistance to P. infestans infection was mediated by the stimulating effect of the B. subtilis 26D and the B. thuringiensis B-6066 bacteria on the concentration of Н2О2, the modulating effect on the activity of hydrolytic enzymes and the enhancement of the transcriptional activity of protease and amylase inhibitor genes in plant tissues. Differences in the degree of activation of the transcriptional activity of hydrolase inhibitor genes by the B. subtilis 26D and the B. thuringiensis B-6066 bacteria were revealed, which suggests differential ways of forming the potato resistance to P. infestans under their influence.


Introduction
One of the ways of increase of plant resistance to pathogenic organisms and adverse environmental factors by ecologically friendly methods is the scientifically substantiated use of non-pathogenic rhizospheric bacteria (PGRP -plant growth promoting rhizobacteria). In this regard, especially attractive are the bacteria of the genus Bacillus, that are highly effective, undemanding to cultivation media, retaining their viability for a long time, and able to activate natural defense mechanisms in a plant organism [1][2].
A lot of compounds have been developed based on Bacillus bacteria in order to protect plants from phytopathogens and pests, the effect of which is based on the natural antagonism of Bacillus to many phytophagous organisms [3]. Thus, the active basis of biological products against a wide range of food crop pests is a complex of protein crystals of delta-endotoxin and thermostable exotoxin of the microbial culture Bacillus thuringiensis var. thuringiensis. The "Fitosporin" compound was created based on the cell culture, metabolites and bacterial spores of Bacillus subtilis 26D, which is used to protect plants from pathogens with various types of parasitism. An important feature of biological products based on living microorganisms is the non-specific activation of plant defense mechanisms [4].
At present, a large amount of literature on development decrease of phytopathogens under the influence of Bacillus bacteria is presented, however, the mechanisms of the induction of plants' protective reactions when treated with these bacteria still remain unclear. The ability of some representatives of the genus Bacillus to multiplicate inside plant tissues is known [5], as well as their ability to secrete hydrolytic enzymes that inhibit the pathogens development [6]. Among the metabolites of Bacillus bacteria, cyclic low molecular weight lipopeptides, such as surfactin and iturin [7] were distinguished, inducing the generation of hydrogen peroxide and an oxylipin signaling system in plants that regulates the activity of proteinase inhibitors [8].
The objective of this work is a comparative study of influence of the Bacillus subtilis Cohn and B. thuringiensis Berliner bacteria on the hydrogen peroxide (Н2О2) content, the activity of hydrolytic enzymes and the transcriptional activity of the genes of hydrolase inhibitors in potato plants during infection with late blight pathogen Phytophthora infestans Mont. de Bary.

Objects of study
We used potato plants grown from micro tubers of the susceptible Early Rose variety. The tubers were planted in containers with soil (TerraVita, peat of varying degrees of decomposition, purified river sand, perlite, complex mineral fertilizer, biohumus; pH 6.0-6.5). Plants were grown on a light site for 15 days, then inoculated with a suspension culture (final titer of 10 8 cells/ml) of B. thuringiensis B-6066 bacteria, obtained from the State Research Institute of Genetics and Selection of Industrial Microorganisms, and B. subtilis 26D bacteria from the commercial biological product "Fitosporin-M" ("Bashinkom", Russia). 5 days after bacteria treatment, the plants were infected with a spore suspension (10 7 spores/ml) of P. infestans -the late blight pathogen. P. infestans (Bashkiria strain) was grown in test tubes on potato-glucose medium for 11 days. In order to obtain zoospores from zoosporangia, the fungus mycelium was poured with distilled water and kept for 30 min at 4 °C, and then 20 min at room temperature. We used the culture of the oomycete Phytophthora infestans (Mont.) de Bary from the collection of the Institute of Biochemistry and Genetics of the Ufa Federal Research Centre of the Russian Academy of Sciences. After 24, 48, and 72 h after infection, the control and infected plants were fixed for the biochemical studies. Bacteria were cultured for 24 hours in Luria-Bertani liquid medium, then the suspension was diluted with distilled water to the required concentration. The development of the disease was observed for 7 days and was estimated by the % ratio of the damage area to the leaf blade area. The leaves were photographed, images were analyzed in the ImageJ program (NIH, USA).

Determination of H2O2 content
The leaves were homogenized in 0.025 M phosphate buffer, pH 6.2 (PB), in a 1:3 ratio, centrifuged for 20 min at 10,000 g in a centrifuge ("Eppendorf", Germany). The supernatant was used to determine the Н2О2 content. The Н2О2 content was evaluated at 560 nm by using xylenol orange. The reagent contained 0.074% Mohr's salt in 5.81% sulfuric acid and 0.009% xylenol orange in 1.82% sorbitol (in a ratio of 1:100). The optical density of the reaction products was measured on a Biospek-Mini spectrophotometer ("Shimadzu", Japan).

Determination of enzymes and inhibitors activity
The activity of proteases, amylases, and inhibitors was determined by hydrolysis of a substrate immobilized in a polyacrylamide gel [9].

Evaluation of the transcriptional activity of potato protease and amylase inhibitor genes
RNA from plants was isolated by using trizol (Molecular Research Center, Inc., USA). In order to obtain cDNA based on the mRNA of the studied samples, a reverse transcription reaction was performed using M-MuLV reverse transcriptase according to the protocol of the supplier. The accumulation of transcripts of the amylase inhibitor genes (GenBank number XM006351484) and the proteinase inhibitor (GenBank number JX683427) was analyzed by real-time quantitative PCR using the iCycler iQ5 Real-Time PCR Detection System (Bio-Rad, USA) and intercalating dye SYBR Green I ("Synthol", Russia). Changes in transcriptional activity of genes (estimation of the number of mRNA copies for each gene) were determined by the St_act relative reference gene ("housekeeping gene", actin, GenBank number X55749) using the iCycler iQ5 Real-Time Detection System software ("Bio-Rad", USA). A computer analysis of amino acid and nucleotide sequences was performed using the Lasergene computer software package from DNASTAR, Inc (USA).

Statistical data processing
The experiments included at least three biological repeats in analysis of the biochemical parameters and at least 15   One of the mechanisms for increasing potato resistance to late blight pathogen P. infestans is associated with a hypersensitivity reaction, which suggests the generation of ROS in plants in response to the pathogen introduction. One of the earliest reactions of plant cells to contact with a pathogen is the production of ROS, which are involved in intracellular signaling through the interconversion.
Studies have shown that inoculation of P. infestans, treatment with the B. subtilis 26D and the B. thuringiensis B-6066 increased the level of Н2О2 in potato plants (Fig. 2). Moreover, treatment with the B. thuringiensis B-6066 bacteria had a more significant stimulating effect on Н2О2 production in healthy plants, and with the B. subtilis 26D -in the infected plants. Hydrogen peroxide is a secondary messenger involved in the work of various plant cell signaling systems and regulating the expression of protective protein genes [10]. By stimulating the accumulation of ROS, endophytic bacteria are likely to induce the transmission of signals forming a defense mechanisms complex. The participation of bacteria Bacillus spp. in the generation of Н2О2 and increasing the resistance of wheat to pathogens of common bunt [11] and Septoria tritici blotch were shown [12].

Influence of genus Bacillus bacteria on activity of hydrolytic enzymes and their inhibitors in potato plants during P. infestans infection
Hydrolytic enzymes of plant pathogens play an important role in their introduction and distribution in the plant organism [13]. Exogenous proteases of microorganisms are involved in the destruction of structural proteins of the cell wall [14] and other plant protective peptides [15], as well as in the processing of their own extracellular proteins of microorganisms necessary for the pathogenesis process [16]. An important role of proteases is indicated by the direct dependence of the intensity of the disease in plants on the activity of microbial proteases observed in a number of cases. The level of proteolytic activity is also associated with necrotic processes. The biological databases contain information on the possibility of P. infestans expression of many proteases of different families: cysteine and serine proteases, proteases of the Hsl, Fts, and Clp families (www.ncbi.nlm.nih.gov/bioproject/PRJNA17665). As the research results showed, infection of plants with P. infestans led to a decrease in the activity of proteases in the potato leaves (Fig. 3a), which could be associated with the activation of their inhibitors (Fig. 5). In healthy and infected potato plants treated with the B. subtilis 26D bacteria, protease activity remained at the level of the corresponding control variant (Fig. 3). When plants were treated with the B. thuringiensis bacteria, there was a slight increase in the activity of proteases in the leaves. The B. subtilis 26D and the B. thuringiensis B-6066 bacteria had a similar effect on the activity of amylases in potato plants (Fig. 3b).
Hydrolytic enzymes that cause destruction of the components of the fungal cell wall are widely represented in bacteria of the genus Bacillus [17]. To date, the presence of various forms of alpha-amylases with molecular masses of 47 kDa (amyA, amyE genes), 60 kDa (amyS gene) and 75 kDa (amyM gene) has been described in the Bacillus bacteria [18][19][20][21][22][23][24]. It can be assumed that phytopathogenic fungi and bacteria release a mixture of hydrolases into the environment, the role of which is both in the destruction of plant structures in order to obtain nutrients, and in active interaction with their protective systems.
Hydrolase inhibitors play an important role in protecting plants against the penetration and spread of phytopathogens [25][26][27]. It is interesting to note that hydrolase inhibitors prevent activity of both microbial enzymes and their own plant enzymes, thereby reducing the level of tissue degradation [28][29]. Infection of potato plants with P. infestans and treatment with the B. subtilis and B. thuringiensis bacteria increased the activity of protease inhibitors in plant tissues (Fig. 4a). For example, activity of protease inhibitors increased 4 times in plants treated with the B. thuringiensis bacteria compared to the control variant (Fig. 4a). Plant protease inhibitors are active against phytopathogenic microorganism enzymes. Thus, protein trypsin inhibitors from potato tubers and legumes can inhibit the exogenous proteinases of the phytopathogenic fungus Rhizoctonia solani Kuhn [30]. High proteolytic activity not only ensures growth and development of the pathogen by amino acids, but also neutralizes the protective proteins of potato -inhibitors of hydrolases and lectins.
A relatively high level of activity of amylase inhibitors was revealed in healthy potato plants, which remained almost unchanged under the influence of treatment with the B. subtilis and B. thuringiensis bacteria (Fig. 4b). P. infestans infection of plants led to a 4-fold decrease in the activity of amylase inhibitors compared to control variant plants. Level of inhibitor activity increased in potato plants that were treated with bacteria, especially under the influence of the B. thuringiensis (Fig. 4b).
Amylases have been shown to be represented by constitutive proteins in most taxonomic groups of fungi [28]. However, in P. infestans amylase is absent. Phytophthora phytopathogens activate the biosynthesis of potato's own amylases in the damaged tissues for breaking down starch [31]. It can be assumed that the decrease in the activity of amylase inhibitors in plants is due to the participation of P. infestans in the inactivation of amylases, which inhibits the growth and development of the pathogen.

Influence of genus Bacillus bacteria on the transcriptional activity of amylase and protease inhibitor genes in potato plants during P. infestans infection
The formation of hydrolase inhibitors in response to pathogen incorporation usually occurs de novo. In this regard, we studied the activity of transcription of genes of protease and amylase inhibitors in potato plants infected with P. infestans and treated with bacteria of the genus Bacillus.
In plants infected with P. infestans and treated with Bacillus bacteria, accumulation of transcripts of the protease inhibitor gene is observed (Fig. 5a). In plant tissues treated with Similar results were obtained when studying the transcriptional activity of the amylase inhibitor gene during treatment with bacteria and P. infestans infection (Fig. 5b). Thus, under the influence of the B. subtilis 26D treatment, the level of transcriptional activity in infected plants increased 2.5 times as compared to the control variant, while the B. thuringiensis B-6066 treated plants showed an increase of 3 times (Fig. 5b). An increase in the transcriptional activity of the protease and amylase inhibitor genes under the influence of B. subtilis and B. thuringiensis, as in uninfected plants, but especially during infection, indicates the sensitivity of these genes to the metabolites of Bacillus spp. bacteria.
It is known that one of the protective mechanisms of plant cells in response to the introduction of pathogenic organisms is the suppression of the activity of secreted exogenous hydrolytic enzymes by specific plant inhibitors. The widespread occurrence of hydrolase inhibitors in plants of various genera and families confirms their important role in ensuring the vital activity of plants. Hydrolase inhibitors play a significant role not only in increasing the resistance of plants to the effects of phytophagous organisms, but also provide regulation of enzyme activity during growth and development of the plant itself. The possibility of inducing the activity of hydrolase inhibitors in plants upon infection by pathogenic organisms seems to be one of the possible mechanisms for controlling their resistance, including by treatment with compounds based on various strains of the genus Bacillus bacteria.

Conclusion
Hence, the obtained research results indicate that the activation mechanism of protective systems in potato plants by endophytic bacteria of the genus Bacillus is mediated by the accumulation of Н2О2 and increased activity of PR proteins. The revealed differences in the activation of transcriptional activity of protease and amylase inhibitor genes under the influence of the B. subtilis 26D and the B. thuringiensis B-6066 bacteria suggest differential ways of resistance formation in potato plants to P. infestans with their participation.